Project-1: Identification of somatic mutations and copy number aberrations that underlie serous endometrial cancers During the previous reporting period, we subjected DNAs from serous endometrial tumors and from the matched normal tissues to whole exome sequencing at the NIH Intramural Sequencing Center. We used a number of steps to filter germline variants (present in both tumor and normal DNAs) from somatic variants (present exclusively in the tumor DNA). We then subjected the somatic variants to orthogonal validation to distinguish true somatic mutations from false-positive calls. Somatic mutations can be causative (driver) mutations or incidental (passenger) mutations. Towards the end of the last reporting period and during the first quarter of the current reporting period we focused our studies on a small number of mutated genes that we hypothesized were driver genes. We resequenced the candidate driver genes from an additional set of primary endometrial tumors to determine their overall mutation frequency and spectrum. Within the present reporting period we initiated functional studies to determine how the mutations in candidate driver genes affect the encoded proteins. In ongoing and planned studies that will extend into the next reporting period, we will: (1) Continue to characterize the function of the mutated proteins. (2) Mine the exome data generated in this study using new algorithms for variant calling that are being developed by our collaborators within NHGRI. (3) Perform a meta-analysis of our exome data combined with the exome data of serous endometrial tumors that is being generated by The Cancer Genome Atlas. (4) Procure additional anonymized, serous tumors and matched normal tissues for the proposed studies. Project 2. Identification of somatic mutations that underlie clear cell endometrial cancer During the current reporting period, we subjected DNAs from clear cell endometrial tumors and from the matched normal tissues to whole exome sequencing at the NIH Intramural Sequencing Center. In ongoing and planned studies that will extend into the next reporting period, we will: (1) Filter germline variants (present in both tumor and normal DNAs) from somatic variants (present exclusively in the tumor DNA). (2) Subject the somatic variants to orthogonal validation using Sanger sequencing and/or mass spectrometry to distinguish true somatic mutations from false-positive calls. (3) Resequence the coding exons of the genes of interest from a larger set of primary endometrial tumors, in a mutation prevalence screen. (4) Prioritize the most significantly gene(s) for subsequent studies to determine the functional consequences of the associated mutations. (5) In parallel, we will continue to procure additional anonymized, clear cell tumors and matched normal tissues for use in the proposed studies. Project 3. Interrogation of somatic copy number alterations in serous and clear cell endometrial cancer In parallel to mutational analyses, we are searching for somatic copy number alterations in serous endometrial tumor genomes. In past reporting periods, we used high-density SNP arrays to catalogue somatic genomic copy number alterations (gains and losses) that occur in serous and clear endometrial tumors. Within the current reporting period, we reanalyzed these data using a refined algorithm. The data reanalysis uncovered several regions of high copy gain and homozygous deletion. In ongoing studies, we narrowed our focus to a set of discrete copy number alterations that occur at high frequency among these tumors. Several focal regions of copy number alteration contain candidate cancer genes or known cancer genes. In ongoing and planned studies that will extend into the next reporting period, we will: (1) Validate the focal copy number alterations using real-time PCR. (2) Determine which gene(s) within the validated region is likely to the target gene(s). (3) Determine the effect of overexpressing or depleting target genes on the growth properties of endometrial cancer cell lines.